Assays that quantify the neovasculature are critical for the development of agents to treat angiogenic diseases like cancer and other vascular diseases that need to stimulate new vessel growth. However, the availability of simple and reliable quantitative assays of angiogenesis has been a consistent limiting factor in angiogenesis research. The long-range goal is to develop a noninvasive detection technology for routine in vivo angiogenesis monitoring in the matrix- implant assay by intravital two-photon fluorescence microscopy or two-photon fluorescence lifetime imaging microscopy. The hypothesis is that with well-designed fluorescent probes with optimized structures possessing high 2PA cross-sections, high fluorescence quantum yield, and both excitation and fluorescence emission at near-IR wavelengths, the depth and quality of the 2PF imaging of angiogenesis may be greatly improved. In the present proposal, the short-term goal is to develop the molecular markers for the in vivo two-photon fluorescence monitoring of angiogenesis. Aim 1. Design and synthesis of highly efficient two-photon absorbing probes as reporters for 2PF and FLIM imaging. Fluorene derivatives will be prepared with greatly improved two-photon absorptivity while maintaining high fluorescence quantum yields and high photostability. In addition, motifs will be introduced to shift the fluorescence emission to wavelengths in the near-IR region for deeper tissue penetration. Aim 2. The optimized 2PF molecules, conjugated with peptides, will target 1v23 integrin. From the studies in Aim 1, probes that satisfy the properties above will be selected for conjugation with peptides, such as RGD, which specifically targets newly grown vessels, to target 1v23 integrin. Aim 3. Determine photophysical properties of the new 2PA angiogenesis probes under linear (single-photon) and nonlinear (two-photon) irradiation conditions. The photophysical properties of the fluorophores to be characterized include single-photon and two-photon absorption and fluorescence spectra, fluorescence excitation anisotropy, fluorescence quantum yield, excited state lifetime, and photostability. The cytotoxicity of the probes will also be investigated to determine if the candidates are biocompatible for in vivo imaging. The future work is to use these materials in the in vivo angiogenesis of tumors together with our collaborators at the M. D. Anderson Cancer Institute Orlando. The results are expected to lead to first demonstration of the continuous monitoring of the matrix-implanted angiogenesis assay. PUBLIC HEALTH RELEVANCE This research aims to provide a new technique and probes for in vivo spatial and temporal monitoring of matrix-implanted angiogenesis assays using less damaging near-infrared light and two-photon fluorescence techniques. The method developed should prove valuable to enable biomedical researchers to study angiogenesis processes in real time without disturbing the tissue. Such a breakthrough will afford a better understanding of disease processes and the mechanism of action of drugs and other species in vivo. [unreadable] [unreadable] [unreadable]